October 18, 2011
Fall 2011
Pool N58, RNA, Lysozyme
Progress,
Results, and Discussion
Lysozyme: Practice Round 1A
Complete
RNA bead selection against lysozyme began on September 1, 2011. The goal of this practice round was to get
acquainted with the bead based selection protocol, learn new lab techniques,
and possibly produce high affinity binders against lysozyme. On September 1, the target lysozyme was
immobilized onto beads and selected against.
I completed three washes (labeled W0-W3), a mix pool binding reaction
(containing RNA from the old N58 pool) and an elution (labeled E1), using 2
volumes of 1X selection buffer for each wash.
On September 6, ethanol precipitation and reverse transcription were performed
on washes W0, W3, and E1. The reactions
were stored in the freezer until September 8, when they were used for cycle
course PCR and gel electrophoresis. No
negative control was made. 5 uL aliquots
of each wash were taken during cycles seven, nine, twelve, fifteen, and twenty
to use for gel electrophoresis. The
results of the gel can be found in Figure
1. The wells were filled according
to the following Table 1:
1.
Ladder
|
7.
Nothing
|
13.
W3- Cycle 7
|
19.
E1- Cycle 7
|
2.
W0- Cycle 7
|
8.
Nothing
|
14.
W3- Cycle 9
|
20.
E1- Cycle 9
|
3.
W0- Cycle 9
|
9.
Nothing
|
15.
W3- Cycle 12
|
21.
E1- Cycle 12
|
4.
W0- Cycle 12
|
10.
Nothing
|
16.
W3- Cycle 15
|
22.
E1- Cycle 15
|
5.
W0- Cycle 15
|
11.
Nothing
|
17.
W3- Cycle 20
|
23.
E1- Cycle 20
|
6.
W0- Cycle 20
|
12.
Nothing
|
18.
Ladder
|
24.
Nothing
|
Figure
1: Lysozyme, N58, Round 1A. The first gel I
ran after cycle course PCR in Round 1A is depicted above and appears to have
been successful. W0, the positive
control, over-amplified as expected because this wash contained all the RNA
strands that did not bind to the target.
Also, E1, which contains potential aptamers against lysozyme, showed
bands before the last wash (W3), suggesting that all possible binders were
collected and all weak or nonbinding proteins were removed by the last
wash. A well defined band for E1 (before
over-amplification) occurred at cycle 12.
E1 for the twelfth cycle is boxed in red above. This clear band for E1 determines the number
of cycles that will be used during large scale PCR. The resolution is not of the best quality
because the gel had to be frozen overnight due to camera malfunctions.
Large scale PCR was then performed on
September 20 to make enough E1 template for transcription. Six 100 uL reactions were made, each
containing E1 from reverse transcription.
A negative control was also made for practice using the same components
for the six reactions (found in the “Bead Based RNA Selection Protocol”), but
instead of using E1, more diH2O was added. These six reactions were run for twelve
cycles as determined by gel electrophoresis after cycle course PCR. On September 30, we combined and ethanol
precipitated the reactions. During this
step, two errors were made. After the reaction
was centrifuged for ten minutes and the supernatant decanted, 300 uL of chilled
70% EtOH was not added to the reaction.
This was not realized until after transcription was underway. The second error was made at the end of
ethanol precipitation. When combing the
pellets of what were initially two 300 uL large scale reactions, 20 uL of diH2O
was accidentally added to each reaction instead of 10 uL. This made the total precipitated dsDNA volume
40 uL instead of 20 uL. Transcription
was then performed on this diluted reaction, and on October 1 a PAGE gel was
run. The results of PAGE appeared to be
successful. Ethanol precipitation was
performed on the gel elution on October 4, but after extended periods of
chilled centrifugation, there was no visible pellet. The gel had not been as successful as
initially assumed, most likely because of the errors during ethanol
precipitation.
Lysozyme:
Practice Round 1B
A second practice round against
lysozyme, also using complete RNA bead selection, began on October 6,
2011. The main objective of this second
practice round was to perfect my selection techniques and complete a full round
by myself. Target immobilization was
performed using buffers I had made the day before, 1X PBS, 10X PBS Selection
buffer with MgCl2, and 1X PBS selection buffer. A mix pool binding reaction was made using RNA
from the new N58 pool. I completed three
washes (labeled W0-W3) and an elution (labeled E1) for this round, using 2
volumes of 1X selection buffer for each wash.
On October 11, they were ethanol precipitated. Each precipitation reaction initially
contained 100 uL of reaction from washes W0, W3, and E1,10 uL 3M NaAc, 3 uL
Glyco-blue, and 1110 uL 100% EtOH. I only
added what was available in the lowest volume wash (W0, ~100 uL), having
forgotten that I had needed to add enough diH2O so that each wash reaction
ended up being 400 uL. I quickly corrected
this so that the new precipitation components were 400 uL of reaction from
washes W0, W3, and E1, 40 uL 3M NaAc, 3 uL Glyco-blue, and 1110 uL 100%
EtOH. Ethanol precipitation was
continued normally. Reverse
transcription was then performed on the precipitated washes. Each reaction totaled 20 uL and their
components can be found in the “Reverse Transcription” section of the “Complete
RNA Bead-Based Selection Protocol.” On
October 13, cycle course PCR, gel electrophoresis, and large scale PCR were
performed. For cycle course PCR, the
same components were used in Round 1A, except this time a negative control was
made using all of the reaction components except for ssDNA from reverse
transcription. diH2O was used
instead. 5 uL aliquots of each wash were
taken during cycles six, nine, twelve, fifteen, and twenty to use for gel
electrophoresis. The results of the failed
gel can be found in Figure 2. The wells were filled according to Table 2:
1.
W0- Cycle 6
|
7.
Ladder
|
13.
Negative Control
|
19.
E1- Cycle 20
|
2.
W3- Cycle 6
|
8.
W3- Cycle 9
|
14.
Ladder
|
20.
Nothing
|
3.
W0- Cycle 9
|
9.
W3- Cycle 12
|
15.
E1- Cycle 6
|
21.
Nothing
|
4.
W0- Cycle 12
|
10.
W3- Cycle 15
|
16.
E1- Cycle 9
|
22.
Nothing
|
5.
W0- Cycle 15
|
11.
W3- Cycle 20
|
17.
E1- Cycle 12
|
23.
Nothing
|
6.
W0- Cycle 20
|
12.
Nothing
|
18.
E1- Cycle 15
|
24.
Nothing
|
Figure
2: Lysozyme, N58, Round 1B. The gel after my
second practice cycle course was a complete failure. There were no apparent bands W0, which should
have the most and earliest over-amplification.
There were also no bands seen under W3, or E-1 until the cycle 20. There appears to be some bands at the very
bottom of the top portion for washes W0 and W3, but bands should not appear
that far down. It is unclear what these
bands are. Also, there is the possibility
of contamination because the negative control, which lacks ssDNA, showed a
band. So what did appear in the well for
E1-Cycle 20 is not reliable, and must not be used to determine the amount of
cycles used for lsPCR. The failure is
possibly due to errors made during reverse transcription.
Although
the gel was a complete failure, I decided to continue on with the round to work
on my selection techniques. Large scale
PCR was performed on the E1 ssDNA from reverse transcription. The components of large scale were the same
as they were for Round 1A. After large
scale, the reactions were placed in the freezer.
Problems
Encountered
Lysozyme:
Practice Round 1A
During the first selection round of
lysozyme, three errors were made. The
first, which may not technically be an error but should be done, was made
during cycle course PCR. A negative
control was not made, so the results that were seen cannot be trusted. There is a possibility that contamination had
occurred, especially since many of the RNA pools were producing bands when used
in no template negative controls.
The second and third errors made
during Round 1A occurred during ethanol precipitation after large scale
PCR. First, chilled 70% EtOH was not
added after the first centrifugation.
This most likely had a major impact on the purification and removal of
dsDNA (made during lsPCR) from the solution.
Not adding this component may have prevented the dsDNA from fully “crashing”
out of the solution. Also, at the end of
ethanol precipitation, twice the amount of diH2O was added than
normal, further diluting the concentration of “crashed” aptamer dsDNA.
Lysozyme: Practice Round 1B
No apparent mistakes had been made
during Round 1B until after cycle course PCR when the gel failed. The exact reason for why the gel failed is
unknown. It was suggested that errors
had to have been made during or right before reverse transcription, and it is
possible that a major component might not have been added to the reverse
transcription reactions. Also, a band
appeared under the negative control, suggesting that there was possible
contamination from the N58 primers.
This, too, had some impact on the outcome of the gel.
Conclusion
and Future Work
The purpose of these two practice rounds was to get
me familiar with the protocol, allow me to quickly acquire new lab skills, and better
understand the research I am doing. My
first two rounds have taken longer than usually, but being new to the stream
this semester, I feel that I have learned a lot and am catching on quickly. I only made a few mistakes during each
practice round, but these mistakes were critical to the development of my
practice project. As stated before, I
will continue on with Round 1B, even though I had a failed cycle course PCR
gel. I plan on completing transcription
and a PAGE gel within the next week. I
will most likely use someone else’s large scale and transcription products
during PAGE, precipitation, and quantitation.
By the end of this week or the beginning of next week, I plan on
starting selection against my real target, BCL-2. By the next report, I plan on completing
three full washes.
A link to my old abstract for DAT can be found here.
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